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De novo truncating variants in ASXL2 are associated with a unique and recognizable clinical phenotype: further involvement of the ASXL gene family in neurodevelopmental genetic syndromes

Vandana Shashi1,31, Loren D.M. Pena1,31, Katherine Kim2, Barbara Burton2, Maja Hempel3, Kelly Schoch1, Magdalena Walkiewicz4, Heather M. McLaughlin5, Megan Cho5, Nicholas Stong6, Scott E. Hickey7, Christine M. Shuss7, UDN Members8, Michael S.Freemark9, Jane S Bellet10, Martha Ann Keels11, Melanie J Bonner12, Maysantoine El-Dairi13, Megan Butler14, Peter G. Kranz15, Constance TRM Stumpel16, Sylvia Klinkenberg17, Karin Oberndorff18, Malik Alawi19,20,21, Rene Santer22, Slavé Petrovski6,23, Outi Kuismin24, Satu Korpi-Heikkilä25, Olli Pietilainen26, Palotie, Aarno27, Mitja I. Kurki28 Alexander Hoischen29, Anna C. Need30, David B. Goldstein6, Fanny Kortüm3

1 Division of Medical Genetics, Department of Pediatrics, Duke Health, Durham, NC, USA 277102 Department of Pediatrics, Lurie Children’s Hospital of Chicago, IL, USA 606113 Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 202464 Baylor College of Medicine, Houston, Texas, USA 770305 GeneDx, Gaithersburg, Maryland, USA 208776 Institute for Genomic Medicine, Columbia University, New York, New York, USA 100327 Division of Molecular and Human Genetics, Nationwide Children’s Hospital, Columbus, OH, USA 430828 Undiagnosed Diseases Network, NIH Common Fund, Bethesda, MD, USA 208929 Division of Endocrinology and Diabetes, Department of Pediatrics, Duke Health, Durham, NC, USA 2771010 Departments of Pediatrics and Dermatology, Duke Health, Durham, NC, USA 2771011 Department of Pediatrics, Duke Health, Durham, NC, USA 27704 12 Psychiatry and Behavioral Sciences, Duke Health, Durham, NC, USA 2771013 Duke Eye Center, Duke Health, Durham, NC, USA 2771014 Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Duke Health, Durham, NC, USA 2771015 Division of Neuroradiology, Department of Radiology, Duke Health, Durham, NC, USA 2771016 Department of Clinical Genetics and School for Oncology &Developmental Biology (GROW), Maastricht University Medical Center, Maastricht, The Netherlands 6202AZ17Department of Neurology Maastricht University Medical Center, Maastricht, The Netherlands 6202AZ18Department of Pediatrics, Zuyderland Medical Center Sittard, the Netherlands 6162 BG19Bioinformatics Service Facility, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2024620Center for Bioinformatics, University of Hamburg, Hamburg, Germany 20246

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21Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Virus Genomics, Hamburg, Germany 2024622Department of Paediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2024623 Department of Medicine, Austin Health and Royal Melbourne Hospital, The University of Melbourne, Melbourne, Victoria, Australia 305024 PEDEGO Research Unit, Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Finland and Department of Clinical Genetics, Oulu University Hospital, Finland, Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland 0029025Northern Ostrobothnia Hospital District, Center for Intellectual Disability Care, Oulu, Finland 9022026Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA, USA, 02142, Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA 0213827Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA 02114 , Program in Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA, 02142, The Stanley Center for Psychiatric Research, The Broad Institute of MIT and Harvard, Cambridge, MA, USA, 02142, Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland 00290, Psychiatric & Neurodevelopmental Genetics Unit, Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA, 02114, Department of Neurology, Massachusetts General Hospital, Boston, MA USA, 0211428Program in Medical and Population Genetics and Genetic Analysis Platform, The Broad Institute of MIT and Harvard, Cambridge, Massachusetts USA 02142,., Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, USA 0211429 Radboud University Medical Center, Department of Human Genetics, Nijmegen The Netherlands 6500HB30Division of Brain Sciences, Department of Medicine, Imperial College London W12 0NN

31Vandana Shashi and Loren Del Mar Pena contributed equally to manuscript

Corresponding Author: Vandana Shashi, MDDivision of Medical Genetics, Department of PediatricsBox 103857, Duke HealthDurham NC 27710Email: vandana.shashi@duke.eduTel: (919) 681-2616

Email Addresses of Authors: vandana.shashi@duke.edu,  loren.pena@duke.edu, kkim@luriechildrens.org, bburton@luriechildrens.org, m.hempel@uke.de,

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kelly.schoch@duke.edu, Magdalena.Walkiewicz@bcm.edu, hmclaughlin@genedx.com, mcho@genedx.com , ns3116@cumc.columbia.edu , michael.freemark@duke.edu, jane.bellet@duke.edu , marthaann.keels@duke.edu , melanie.bonner@duke.edu, mays.el-dairi@duke.edu , megan.butler@duke.edu , peter.kranz@duke.edu, c.stumpel@mumc.nl, s.klinkenberg@mumc.nl, k.oberndorff@zuyderland.nl, m.alawi@uke.de, r.santer@uke.uni-hamburg.de , slavep@unimelb.edu.au , outi.kuismin@oulu.fi, satu.korpiheikkila@ppshp.fi,olli.pietilainen@helsinki.fi, aarno.palotie@helsinki.fi, mitja.kurki@gmail.com, Alexander.Hoischen@radboudumc.nl, a.need@imperial.ac.uk, DG2875@columbia.edu, f kortuem@uke.de

Keywords: ASXL2, Macrocephaly, Whole exome sequencing. Developmental/Intellectual Disabilities, Glabellar nevus flammeus

Word count (excluding title page, abstract, references, figures and tables): 2515

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ABSTRACT

The ASXL genes (ASXL1, ASXL2 and ASXL3) participate in body patterning during

embryogenesis and encode for proteins that are involved in epigenetic regulation and assembly

of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1

and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers

syndromes, resulting in overlapping features of severe intellectual disability and dysmorphic

features. ASXL2 has not yet been associated with a human Mendelian disorder.

Whole-exome sequencing was performed in six unrelated probands with developmental delay,

macrocephaly and dysmorphic features. All six had de novo truncating variants in ASXL2. A

careful review enabled the recognition of a specific phenotype consisting of macrocephaly,

prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding

difficulties, hypotonia and developmental disabilities. Although overlapping features with

Bohring-Opitz syndrome and Bainbridge-Ropers syndromes exist, features that distinguish the

ASXL2-associated condition from ASXL1 and ASXL3 disorders are macrocephaly, absence of

growth retardation and more variability in the degree of intellectual disabilities.

We were also able to demonstrate with mRNA studies that these variants are likely to exert a

dominant negative effect, since both alleles are expressed in blood, with the mutated ASXL2

transcripts escaping nonsense mediated decay. In conclusion, de novo truncating variants in

ASXL2 underlie a neurodevelopmental syndrome, with a clinically recognizable phenotype. This

report expands the germline disorders that are linked to the ASXL genes.

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REPORT

The three additional sex combs-like genes (ASXL1, 2 and 3) code for polycomb proteins that act

as histone methyltransferases1, serve as epigenetic scaffolding proteins, and are involved in body

patterning2. Somatic mutations of all three ASXL genes occur across a range of malignancies 3.

Drosophila Asxl genes are involved in homeotic gene activation and silencing4; 5. In mice, Asxl2

has been reported to regulate skeletal, lipid and glucose homeostasis and cardiac development6; 7.

Additionally, Asxl1 and Asxl2 fine-tune adipogenesis in mice, with Asxl2 promoting

adipogenesis and Asxl1 inhibiting it8; 9. Homozygous Asxl2 knockout mice demonstrate

premature death, growth retardation, impaired cardiac function and vertebral abnormalities,

indicating that it is required for embryonic and postnatal development5.

Germline mutations of ASXL1 (MIM 612990) and ASXL3 (MIM 615115) have been associated

with specific genetic syndromes10. Truncating variants in ASXL1 cause Bohring-Opitz syndrome

(MIM 605039), a severe disorder with growth retardation, microcephaly, profound intellectual

disability, nevus flammeus of the face, flexion of elbows and wrists and ulnar deviation of the

hands 11; 12. ASXL3 germline truncating variants are associated with Bainbridge-Ropers syndrome

(MIM 615485), characterized by severe intellectual disability, growth retardation, and clinical

features overlapping Bohring-Opitz syndrome 13. Variants in ASXL3 have also been reported in

autism spectrum disorder 14. In contrast, ASXL2 (MIM 612991) has thus far not been implicated

in human Mendelian disease. One individual with a t(2;9) translocation that resulted in a fused

transcript of ASXL2 and KIAA1803 had a complex phenotype of agenesis of the corpus callosum,

ocular colobomas and periventricular heterotopias15, but the relative contributions of the two

genes to this individual’s phenotype are unclear. Similarly, the DECIPHER database lists five

individuals with cytogenetic deletions encompassing ASXL2 and other genes (n=35 to 141), with

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phenotypes that include developmental delays/intellectual disabilities, among other

manifestations (Table S1). Due to the contiguous deletion of several other genes, the specific

contribution of the ASXL2 deletion to these phenotype cannot be determined. An additional

individual with a single de novo base pair deletion is also included, with head, neck, nervous,

skeletal, skin and respiratory system abnormalities, with potential overlapping manifestations

detailed in our individuals. In this study, we present six unrelated individuals ranging from

11 months to 31 years of age with de novo heterozygous truncating variants in ASXL2 detected

by whole-exome sequencing (WES). All individuals share overlapping clinical features including

developmental/intellectual impairments, macrocephaly, distinct facial dysmorphisms, facial

nevus flammeus, feeding difficulties in the newborn period and hypotonia (Table 1 and Figure

1). Detailed clinical summaries are available in the Supplemental Data: Case reports.

Five of the individuals (individuals 1-3, 5-6) underwent trio whole-exome sequencing (WES)

whereas for individual 4 only the individual was sequenced with Sanger sequencing of ASXL2 on

the individual and the biological parents. WES was performed after written informed consent

was obtained through respective Institutional Review Boards and Ethics Committees.

Experienced pediatricians and geneticists clinically assessed the individuals.

For all six individuals, DNA was extracted from maternal, paternal, and proband blood samples.

The exome was captured with Biotin-labeled VCRome 2.1 in-solution Exome probes

(individuals 1 and 4), Agilent Clinical Research Exome Kit (individual 2), Nextera® Rapid

Capture Exome Kit (individuals 3 and 6), or Agilent SureSelect v4 (individual 5) and the exomes

were sequenced on the Illumina platforms HiSeq2000 (individuals 2 and 5) or 2500 (individuals

1, 3, 4 and 6). Two paired-end 100 bp reads were used for the exome-capture sequencing. For

individual 3, Trimmomatic16 was employed to remove adapters, low quality (Phred quality score

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< 5) bases from the 3' ends of sequence reads. Reads shorter than 36bp were subsequently

removed.

The sequencing methodology, further processing and variant interpretation protocols have been

described previously 17-19 20; 21. Functional annotation and alterations filtering in all cases were

performed against public databases (EVS, dbSNP138, 1,000Genomes, ExAC – see Web

Resources). Quality metrics for all WESs are in Table S2. GeneMatcher a web-based tool for

researchers and clinicians working on identical genes, connected the investigators from the

different institutions22.

Processing of the whole-exome data on the six unrelated individuals with overlapping clinical

features (Figure 1 and Table 1) and the healthy parents of five of them, to identify genes having

functionally relevant variants (new, clinically associated, or of low or unknown frequency), took

into account X-linked, autosomal recessive and autosomal dominant inheritance models (Table

S3). We identified one to two putatively de novo variants in the five trios, which were not

present in any variant database (Table S3). De novo variants (frameshift and stop-gained) in

ASXL2 were detected among all individuals (Table 1): Individual 1 – c.2424delC [p.

(Thr809Profs*32)], Individual 2 –: c.2081dupG [p.(Gly696Argfs*11)],)], Individual 3 –:

c.1225_1228delCCAA [p.(Pro409Asnfs*13)], Individual 4 –: c.2472delC [p.(Ser825Valfs*16)],

Individual 5 –: c.2971_2974delGGAG, p.(Gly991Argfs*3), Individual 6 –: c.1288G>T [p.

(Glu430*)] (Mutation designations refer to transcript NM_018263.4 and Build GRCh37/hg19).

*)]. All ASXL2 variants were validated by Sanger sequencing and the de novo origin confirmed

with parental segregation studies (Figure S2A). Interestingly, all identified variants locate to the

penultimate or last exon of ASXL2 (Figure 2).

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Examination of the ExAC database (release 0.3) for loss of function (LoF) variants that passed

ExAC’ s variant quality thresholds revealed two distinct frameshift, two distinct nonsense, and

two distinct canonical splice variants, all with only a single carrier observation (p.Lys668*,

p.Ala658Glyfs*49, p.His632Serfs*17, p.Ser467*, c.505-1G>C and c.940-2A>G). Except for

c.505-1G>C and c.940-2A>G, the ExAC variants are located in a region between the variants

found in Individuals 2 and 6, upstream of the variants in Individuals 1, 4 and 5 and downstream

of the variant found in Individual 3 (Figure 2). The annotation of these LoF variants in ExAC

prompted us to calculate the probability of finding ASXL2 variants by chance in exomes for

neurodevelopmental phenotypes: the six ASXL2 truncating variants in our individuals occurred

in a collective patient base of 12,030, ascertained for neurological and neurodevelopmental

disorders (GeneDx=3,677, University Medical Center Hamburg=123, Baylor=6,198, Radboud

University Medical Center= 1,866, and Broad Institute=166). We used the CRAN function

denovolyzeR23 package with the parameters set for six truncating variants in ASXL2, and

estimated that the probability for the chance occurrence of the six de novo ASXL2 truncating de

novo mutations among 12,030 individuals is 1.47e-10. Correcting this for the 18,668 protein-

coding genes present in the consensus coding sequence (CCDS release 14), this observation is

significant genome-wide (2.744196e-06). Conversely, for the probability of finding six ASXL2

truncating variants in the ExAC database of 60,000 samples, a p value of 1.79e-06 is obtained,

which corrected for 18,668 genes is 0.03341572. These data indicate that there are less than

expected truncating ASXL2 variants within the control database as well. ASXL2 is also highly

intolerant to LoF variants (probability of 0.99), based on the ExAC pLI calculation24. The ExAC

database endeavors not to include subjects with severe pediatric disease, but is enriched for

adults with heart and metabolic diseases, cancer, schizophrenia, and Tourette syndrome24;

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Interestingly, truncating variants in ExAC controls have been reported for both ASXL1 and

ASXL3; these overlap the disease-associated variants, occurring within the last two exons of both

genes 13; 25. Similarly, other genes involved in intellectual disability such as ARID1B have also

been found to have truncating variants in ExAC controls25. The exact explanation for these ExAC

variants that overlap the disease-associated variants for the three ASXL genes remains unclear,

but possibilities include somatic mosaicism, variable expressivity depending on location of the

variants in the gene, or reduced penetrance. The finding that clonal hematopoiesis in healthy

subjects results in acquired somatic variants in genes (including ASXL1) that are also mutated in

myeloid cancers supports the possibility that the variants in ExAC could be somatic rather than

germline24. Options such as examining read counts to determine if a variant could be post-zygotic

are subject to errors due to sequencing factors such as variability in capture, and so at this time

definite inferences cannot be made about the origin of these variants. Non-penetrance would be a

less likely explanation, given the severity of the germline ASXL disorders, but a recent report

confirmed that incomplete penetrance of Mendelian diseases is likely to be more common than

realized26. Future studies on all three ASXL genes are necessary to understand better the overlap

between truncating variants in controls and affected individuals.

Similar to ASXL1 and ASXL3, we initially postulated that haploinsufficiency would be

responsible for the ASXL2 phenotype; alternatively, a dominant negative mechanism could cause

disease. When truncating variants preferentially accumulate towards the end of a gene, the

resulting transcript may escape nonsense mediated decay (NMD) and interfere with the wildtype

protein, causing an abnormal phenotype. The truncating variants in our six cases are within the

last two exons of ASXL2, similar to the distribution of disease variants in ASXL1 and ASXL3 11; 13;

25. To get an idea whether haploinsufficiency or a dominant negative effect could be the

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underlying mechanism of disease, we performed cDNA studies. Fresh venous blood samples of

individuals 1, 3 and 4 were obtained into PAXgene Blood RNA Tubes (PreAnalytiX; QIAGEN,

Hilden, Germany) and total RNA was extracted by use of the PAXgene Blood RNA Kit

(QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. 1 μg of RNA

from each individual was reverse transcribed into cDNA (Superscript III®; Invitrogen,

Karlsruhe, Germany) using random hexanucleotides (Invitrogen, Karlsruhe, Germany) according

to the manufacturer's protocol. PCR amplification for allele expression analysis of ASXL2 was

carried out utilizing Advantage cDNA PCR Kit (Clontech, Takara Bio, Saint-Germain-en-Laye,

France) and primers spanning exon-exon-boundaries of ASXL2. For Individuals 1 and 4, primers

ASXL2_RT_11for (5′-CGACAAGAGATTGAGAAGGAG-3′) located in exon 11 and

ASXL2_RT_12rev (5′-CCATCAGCTGCACAATGAA-3′) located in exon 12 were used; for

Individual 3, primers ASXL2_RT_10for (5′-TGGAAAGAACAATTCTTTGAAAG-3′) and

ASXL2_RT_11rev (5′-CACTCTGACTGGGAGACTACTG-3′) located in exon 10 and exon 11,

respectively, were used. Three temperature ‘touchdown’ PCR was performed using an initial

annealing temperature of 60°C, which was decreased twice by 2°C after three cycles each, then

maintained at 56°C for 29 more cycles. Each cycle consisted of a denaturation step - 30 s at

94°C, an annealing step for 30s, and a 1 min, 68°C elongation. ASXL2_RT_12seq (5’-

TCATAGTGTCTCTGGA-3’) was used for sequencing of PCR products of Individuals 1 and 4.

We found that both wild-type and mutant transcripts were detectable in equal levels in all

investigated individuals (Figure S2B). This suggested to us that at least for the three analyzed

ASXL2 mutations the mRNA containing the respective mutation is unlikely to undergo NMD,

thus providing support for a dominant negative rather than haploinsufficiency as the mechanism

of disease. Similarly, the mechanism of disease with ASXL1 and ASXL3 needs further

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clarification. Initial evidence was presented by Bainbridge to indicate that in a cell line of an

individual with an ASXL3 truncating variant both alleles were expressed, suggesting that the

truncating variant escaped NMD13. In contrast, others have demonstrated that transcripts from

truncating variants in ASXL3 were indeed subject to NMD, making a dominant negative effect

less likely. Nonetheless, since NMD is a variable process, depending on the tissue and time in

development, this remains a possibility in ASXL related disorders27. Additional evidence for a

dominant negative/gain of function effect of truncating ASXL1 variants in cancers28 emphasizes

the importance of further functional studies across all the ASXL genes to understand mechanisms

of disease.

The recognizable pattern of features and the corresponding human phenotype ontology (HPO)

terms seen in our individuals include macrocephaly (HP:0000256), a typical facial appearance

consisting of glabellar nevus flammeus (HP:0001052), hypertelorism (HP:0000316), arched

eyebrows (HP:0002553), prominent eyes, broad nasal tip (HP:0000455) and minor ear

abnormalities. Minor congenital heart disease (HP:0030680), neonatal feeding difficulties

(HP:0011968), hypotonia (HP:0001252), seizures (HP:0001250), brain MRI findings suggestive

of cerebral atrophy (Figure S1) are also consistently seen as well as developmental delays

(HP:0001263) and intellectual disabilities (HP:0001249) ranging from low average cognition to

severe impairments. Less consistent features include multiple capillary malformations and

skeletal findings. Persistent hypoglycemia occurred in Individuals 2 and 3, of unclear cause. The

cause of hypoglycemia in both cases is not clear but the observation of macrosomia at birth and

variably in later life, the inappropriately low free fatty acids and ketones and high insulin

concentration in Individual 3 on one occasion suggests that an impaired regulation of insulin

production or a disturbed peripheral effect on adipose tissue cells may play a role in this.

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Germline damaging variants in ASXL1 and ASXL3 cause Bohring-Opitz and Bainbridge-Ropers

syndromes, respectively11; 13. Overlapping features between our individuals and those with these

disorders include developmental delays, arched eyebrows, feeding difficulties and prominent

eyes. A glabellar nevus flammeus is a striking feature in our six individuals and is seen in the

majority of individuals with Bohring-Opitz syndrome, but not in Bainbridge-Ropers syndrome.

Features that distinguish our individuals from these disorders include macrocephaly, normal

height and weight, absence of a characteristic positioning of the hands and more variability in the

degree of cognitive impairments. The overlap between the manifestations in the three different

disorders may be due to the similarities in function between the members of the ASXL family of

genes. Conversely, the distinctive features of the ASXL2 associated disorder could be attributed

to the differences in its function relative to the other two ASXL genes8. Overall, the presence of a

glabellar nevus flammeus with developmental disabilities should prompt consideration of an

ASXL-related disorder.

ASXL proteins function as tumor suppressors or oncogenes and truncating somatic variants of all

three ASXL genes are found in many malignancies3; 29. Interestingly, Wilms tumor and

nephroblastomatosis has been described in individuals with Bohring-Opitz syndrome30; 31. Thus

surveillance for Wilms tumor has been recommended for individuals with Bohring-Opitz

syndrome30. It is unknown at this time if germline damaging variants in ASXL2 would confer a

risk of cancer; while none of our individuals has had a malignancy, clinical surveillance would

be important in follow-up.

In Asxl2 -/- mice, fetuses have reduced body weight, skeletal anomalies, and cardiac dysfunction

with enlarged hearts and premature death5. ASXL2 is also ubiquitously expressed in the brain of

mouse embryos5 and is reported to regulate bone mineral density, being responsible for genesis

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of osteoclasts, as well as for bone resorption 6; 32. Asxl2-/- mice have been variably reported as

having decreased bone density or osteopetrotic bone. Interestingly, Individual 1 in our series has

been noted to have increased alveolar bone density, a highly unusual dental finding. Individual 4

has decreased bone density with fractures and Individual 6 has mild osteoporosis; it is unclear at

this point if mineralization could be abnormal in other individuals with this ASXL2 related

disorder; more clarity on this should become available with more reports of individuals with

ASXL2 truncating variants.

ASXL2 plays an important role in heart morphogenesis and the transition from fetal to postnatal

circulation33 . Asxl2-/- mice display congenital heart malformations including thickened compact

myocardium in the left ventricle, membranous VSD and dilated cardiomyopathy 5. Four of the

six individuals in our report had minor congenital heart disease, suggesting that cardiac

malformations may be common in this condition.

In conclusion, we have identified a syndrome characterized by developmental disabilities,

macrocephaly and dysmorphic features attributable to germline truncating variants in ASXL2.

Further clinical reports of individuals with ASXL2 damaging variants and related clinical features

as well as functional investigations will reveal the full phenotype of this syndrome.

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Web Resources:

The URLs for data presented herein are as follows:

1000 Genomes Project, http://www.1000genomes.org/

Combined Annotation Dependent Depletion, CADD, http://cadd.gs.washington.edu/score

CRAN packages, https://www.r-project.org/

dbSNP, http://www.ncbi.nlm.nih.gov/SNP/

DECIPHER, https://decipher.sanger.ac.uk

Ensembl Variant Effect Predictor 83, http://ensembl.org/info/docs/tools/vep/index.html

Exome Aggregation Consortium, ExAC, http://exac.broadinstitute.org/

Human Phenotype Ontology Browser, HPO,

http://www.human-phenotype-ontology.org/hpoweb?id=HP:0000118

LoFTEE, https://github.com/konradjk/loftee/

MutationTaster, http://www.mutationtaster.org/

NHLBI Exome Sequencing Project (ESP) Exome Variant Server,

http://evs.gs.washington.edu/EVS/

OMIM, http://www.omim.org/

PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/

SIFT, http://sift.jcvi.org/www/SIFT_enst_submit.html

Trimmomatic, http://www.usadellab.org/cms/?page=trimmomatic

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Consortium: Undiagnosed Diseases Network Members (UDN) Carlos A Bacino cbacino@bcm.eduBrendan H Lee blee@bcm.eduAshok Balasubramanyam ashokb@bcm.eduLindsay C Burrage burrage@bcm.eduGary D Clark gdclark@texaschildrens.orgWilliam J Craigen wcraigen@bcm.eduShweta U Dhar dhar@bcm.eduLisa T Emrick emrick@bcm.eduBrett HGraham bgraham@bcm.eduMahimJain mahimj@bcm.eduSeema R Lalani seemal@bcm.eduRichard A Lewis rlewis@bcm.eduPaolo M Moretti pmoretti@bcm.eduSarah K Nicholas sknichol@bcm.eduJordan S Orange orange@bcm.eduJennifer E Posey Jennifer.Posey@bcm.eduLorraine Potocki lpotocki@bcm.eduJill A Rosenfeld mokry@bcm.eduDaryl A Scott dscott@bcm.eduNeil A Hanchard hanchard@bcm.eduTran A Alyssa alyssat@bcm.eduAlejandro E Mercedes mercedes@bcm.eduAzamian S Mashid azamian@bcm.eduHugo J Bellen hbellen@bcm.eduShinya Yamamoto yamamoto@bcm.eduMichael F Wangler mw147467@bcm.eduMonte Westerfield monte@uoneuro.uoregon.eduJohn H Postlethwait jpostle@uoneuro.uoregon.eduChristine M Eng ceng@bcm.eduYaping Yang yapingy@bcm.eduDonna M Muzny donnam@bcm.eduPatricia A Ward pward@bcm.eduRachel B Ramoni rachel_ramoni@hms.harvard.eduAlexa T McCray alexa_mccray@hms.harvard.eduIssac S Kohane isaac_kohane@hms.harvard.eduIngrid A Holm ingrid.holm@childrens.harvard.eduMatthew Might matthew_might@hms.harvard.eduPaul Mazur paul_mazur@hms.harvard.eduKimberly Splinter kimberly_splinter@hms.harvard.eduCecilia Esteves cecilia_esteves@hms.harvard.edu

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Vandana Shashi vandana.shashi@duke.eduYong-hui Jiang yong-hui.jiang@dm.duke.eduLoren DM Pena loren.pena@dm.duke.eduAllyn McConkie-Rosell allyn.mcconkie@duke.eduKelly Schoch kelly.schoch@duke.eduRebecca C Spillmann rebecca.crimian@duke.eduJennifer A Sullivan jennifer.sullivan@duke.eduNicole M Walley nicole.walley@duke.eduDavid B Goldstein dg2875@columbia.eduNicholas Stong ns3116@cumc.columbia.eduAlan HBeggs beggs@enders.tch.harvard.eduJoseph Loscalzo jloscalzo@partners.orgCalum A MacRae camacrae@bics.bwh.harvard.eduEdwin K Silverman ed.silverman@channing.harvard.eduJoan MStoler joan.stoler@childrens.harvard.eduDavid A Sweetser dsweetser@partners.orgRichard L Maas maas@genetics.med.harvard.eduJoel B Krier jkrier@partners.orgLance H Rodan lance.rodan@childrens.harvard.eduChris A Walsh Christopher.Walsh@childrens.harvard.eduCynthia M Cooper CCOOPER1@mgh.harvard.edJuan C Pallais Juan.Pallais@mgh.harvard.eduLaurel A Donnell-Fink LDONNELL-FINK@PARTNERS.ORGElizabeth L Krieg EKRIEG@PARTNERS.ORGSharyn A Lincoln Sharyn.Lincoln@childrens.harvard.eduLauren C Briere LBRIERE@PARTNERS.ORGHoward J Jacob hjacob@hudsonalpha.orgElizabeth A Worthey lworthey@hudsonalpha.orgJoe Lazar jlazar@hudsonalpha.orgKim A Strong kstrong@hudsonalpha.orgLori H Handley lhandley@hudsonalpha.orgJ. ScottNewberry snewberry@hudsonalpha.orgDavid P Bick dbick@hudsonalpha.orgMolly C Schroeder mschroeder@hudsonalpha.orgDonna M Brown dbrown@clinicallab.orgCamille L Birch cbirch@clinicallab.orgShawn E Levy slevy@hudsonalpha.orgBradenE Boone bboone@hudsonalpha.orgDan C Dorset ddorset@hudsonalpha.orgAngelaL Jones ajones@hudsonalpha.orgTeri A Manolio manolio@nih.gov

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John J Mulvihill John.Mulvihill@nih.govAnastasia L Wise anastasia.wise@nih.govJyoti GDayal jyotig@mail.nih.govDavid J Eckstein david.eckstein@nih.govDonna M Krasnewich dkras@mail.nih.govCarson R Loomis loomisc@mail.nih.govLaura A Mamounas mamounas@ninds.nih.govBrenda Iglesias brenda.iglesias@nih.govCasey Martin casey.martin@nih.govDavid M Koeller koellerd@ohsu.eduThomas O Metz thomas.metz@pnnl.govEuan A Ashley Euan@stanford.eduPaul G Fisher pfisher@stanford.eduJonathan A Bernstein Jon.Bernstein@stanford.eduMatt T Wheeler wheelerm@stanford.eduPatricia A Zornio pzornio@stanford.eduDaryl M Waggott dwaggott@stanford.eduAnnika M Dries annikamd@stanford.eduJennefer N Kohler jkohler@stanfordhealthcare.orgKatrina M Dipple Kdipple@mednet.ucla.eduStan F Nelson snelson@ucla.eduChristina GS Palmer cpalmer@mednet.ucla.eduEric Vilain evilain@ucla.eduPatrick Allard pallard@ucla.eduEsteban C Dell Angelica edellangelica@mednet.ucla.eduHane Lee hanelee@mednet.ucla.eduJanet S Sinsheimer janet@mednet.ucla.eduJeanette C Papp jcpapp@mednet.ucla.eduNaghmeh Dorrani ndorrani@mednet.ucla.eduMatthew R Herzog mherzog@mednet.ucla.eduHayk Barseghyan haykbarseghyan@ucla.eduDavid R Adams david.adams@nih.govChristopher J Adams christopher.adams@nih.govElizabeth A Burke elizabeth.burke2@nih.govKatherine R Chao katherine.chao@nih.govMariska Davids mariska.davids@nih.govDavid D Draper draperd@mail.nih.govTyra Estwick tyra.estwick@nih.govTrevor S Frisby trevor.frisby@nih.govKate Frost kate.frost@nih.govWilliam A Gahl gahlw@mail.nih.gov

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ValerieGartner valerie.gartner@nih.govRena A Godfrey godfreyra@mail.nih.govMitchell Goheen mitchell.goheen@nih.govGretchen A Golas gretchen.golas@nih.govMary G Gordon gracie.gordon@nih.govCatherine A Groden catherine.groden@nih.govAndreaL Gropman agropman@cnmc.orgMary E Hackbarth mary.hackbarth@nih.govIsabel Hardee isabel.hardee@nih.govJean MJohnston johnstonjm@mail.nih.govAlanna E Koehler alanna.koehler@nih.govLea Latham lea.latham@nih.govYvonne L Latour yvonne.latour@nih.govChyau Yueh C Lau christopher.lau@nih.govPaul R Lee paul.lee@nih.govDenise J Levy denise.levy@nih.govAdam P Liebendorder adam.liebendorfer@nih.govEllen FMacnamara ellen.macnamara@nih.govValerieV Maduro vbraden@mail.nih.govMay V Malicdan maychristine.malicdan@nih.govThomas C Markello markellot@mail.nih.govAlexandra J McCarty alexandra.mccarty@nih.govJennifer L Murphy jennifer.murphy@nih.govMichele E Nehrebecky michele.nehrebecky@nih.govDonna Novacic donna.novacic@nih.govBarbara N Pusey barbara.pusey@nih.govSarah Sadozai sarah.sadozai@nih.govKatherine E Schaffer katherine.schaffer@nih.govPrashant Sharma prashant.sharma@nih.govAriane G Soldatos ariane.soldatos@nih.govSara P Thomas sarah.thomas@nih.govCynthia J Tifft ctifft@nih.govNathanial J Tolman nate.tolman@nih.govCamiloToro toroc@mail.nih.govZaheer M Valivullah zaheer.valivullah@nih.govColleen E Wahl colleen.wahl@nih.govMike Warburton mike.warburton@nih.govAlec A Weech alec.weech@nih.govLynne A Wolfe lynne.wolfe@nih.govGuoyun Yu guoyun.yu@nih.govRizwan Hamid rizwan.hamid@vanderbilt.edu

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John H Newman john.newman@vanderbilt.eduJohn A Phillips John.a.phillips@vanderbilt.eduJoy D Cogan joy.cogan@vanderbilt.edu

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Acknowledgements:

We are grateful to the patients and their families who contributed to this study and allowed us to publish their information and pictures.

We thank I. Jantke for skillful technical assistance.

Please see attached conflict of interest form for that information

Conflicts of Interest: The following authors have disclosed the following:

Magdalena Walkiewicz: The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from molecular testing offered at the Baylor Genetics Laboratories.David Goldstein has received consultancy fees and has equity ownership in Pairnomix, LLC. Heather McLaughlin and Megan Cho: employees of GeneDx, Gaithersburg, MD. Barbara Burton has received honoraria, consulting fees or funding for clinical trials from Sanofi- Genzyme, Shire, Biomarin, Ultragenyx, Alexion, Armagen and ReGenX Bio. Eaun Ashley is a Co-Founder of Personalis, Inc.

Funding sources:

This work was supported by: Undiagnosed Diseases Network (1U01HG007672-01 to Shashi V) The Deutsche Forschungsgemeinschaft (KO 4576/1-1 to Kortüm F)

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Appendices: None

Description of Supplemental Data: Case reports, Figure S1, Figure S2, Table S1, Table S2, Table S3 is in this section

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Figure Legends:

Figure 1: Clinical photographs of four patients with de novo ASXL2 mutations.

(A-C) Individual 1 at age 3 years (A) and 8.5 years (B and C). Epicanthal folds with a wide nasal bridge, arched eyebrows, ptosis of eyelids, prominent eyes, hypertelorism, broad nasal tip and V-shaped glabellar nevus flammeus are evident, along with capillary malformation on neck and shoulder (C). (D-F) Individual 2 at six weeks of age (D), at 5 months (E), and at age 10 months (F). Note large glabellar nevus flammeus, thick and arched eyebrows with synophrys, proptosis of the eyes, hypertelorism, epicanthal folds, broad nasal tip and retrognathia. (G-I) Individual 3 presented at the age of 10 month (G) and 4 years (H, frontal view, and I, lateral view) with a flat face (I), broad forehead, prominent glabella, glabellar nevus flammeus, hypertelorism, synophrys, arched eyebrows, ptosis, down slanting palpebral fissures, broad nasal tip, long philtrum, small upper vermilion and small mouth. (J-L) Individual 4 after birth (J), at age 20 month (K) and 3 years (L) with proptosis, small mouth, arched eyebrows, and glabellar nevus flammeus. Individual 5 at age 3 months (M) and at 7 years 10 months (N-O). Note the glabellar nevus flammeus, prominent eyes, hypertelorism, arched eyebrows and broad nasal tip. Individual 6 at age 6 years (P) and 16 years, (Q-R), demonstrating the hypertelorism, likely macrocephaly, broad nasal tip and the glabellar nevus flammeus.

Figure 2: Schematic protein structure of ASXL2. The ASXN, ASXM, and PHD-type Zinc finger domains, which are conserved throughout the ASXL family, are represented as light grey boxes and the LVTQLL motif, a nuclear receptor binding motif, is indicated in dark grey. The location of all domains and motifs is given underneath by amino acid (AA) position as well as a schematic representation of the exons encoding the above depicted part of the protein. The positions of the de novo mutations identified in this study are marked with vertical, red arrows. Small green arrows point to positions of known heterozygous variants (stop gained, frameshift, and splice site) found in apparently healthy individuals (from ExAC database, http://exac.broadinstitute.org/).

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Table 1: Clinical features of four patients with ASXL2 variants, compared to features in ASXL1 and ASXL3 associated disorders

Individual 1 Individual 2 Individual 3 Individual 4 Individual 5 Individual 6 Bohring-Opitz Bainbridge-Ropers

Gene ASXL2 ASXL1 ASXL3Mutation* c.2424delC p.

(Thr809Profs*32)c.2081dupGp.(Gly696Argfs*11)

c.1225_1228delCCAAp.(Pro409Asnfs*13)

c.2472delC p.(Ser825Valfs*18)

c.2971_2974delGGAGp.(Gly991Argfs*3)

c.1288G>Tp.(Glu430*)

Truncating Truncating

Prenatal Findings

None None None None Left renal agenesis,cerebral ventriculo-megaly, intrauterine growth restriction

None IUGR, Polyhydramnios

IUGR

Birth growth parameters

Height, weight >97th

percentile, OFC 92nd percentile

Height, weight and OFC >97th percentile

Height 39th percentile, length >97th percentile, OFC 91st percentile

Weight 35th percentile (length and OFC not available)

Weight 2nd percentile,length 23rd percentile, (OFC not available)

Weight 30th percentile, length 60th percentile, OFC 70th percentile

SGA SGA

Growth parameters at last examination

At 8 years height, weight, and OFC 100th percentile (macrocephaly)

At 10 months height and weight normal, OFC 96th percentile (macrocephaly)

At 5 years height and weight normal, OFC >97th percentile (macrocephaly)

At 4 years height and weight normal, OFC >97th percentile (macrocephaly)

At 7 years 10 months height 99th percentile, weight 92nd percentile, OFC 99nd percentile(macrocephaly)

At 12.8 years weight 88thth percentile, height 1st percentile, OFC 80th percentile (relative macrocephaly)

Severe growth retardation, microcephaly

Severe growth retardation, microcephaly

Feeding difficulties

Present, only shortly after birth

Present Present, only in neonatal period

Present, only shortly after birth

Present for several weeks after birth

Present, only shortly after birth

Present, persistent Present, severe

Hypotonia Present Present, persistent Present, but hypertonia in limbs

Present Present Present Present Present

DD/ID Low average cognition

Moderate Severe Severe Borderline Moderate Severe Severe

Seizures Febrile None Febrile and non-febrile Febrile Suspected Present Present NAFacial features

Hypertelorism, arched eyebrows, long face, prominent eyes, ptosis of eyelids, epicanthal folds, broad nasal tip

Hypertelorism, arched eyebrows, proptosis, epicanthal folds, long eyelashes, synophrys, broad nasal tip

Hypertelorism, arched eyebrows, prominent eyes, ptosis of eyelids, epicanthal folds, prominent glabella, synophrys, small upper vermilion, broad nasal tip

Hypertelorism, arched eyebrows, long face, proptosis, small mouth

Hypertelorism, arched eyebrows, prominent eyes, long eyelashes, small upper vermilion, broad nasal tip

Hypertelorism, malar hypoplasia, low nasal bridge, short philtrum, ptosis, broad nasal tip, high narrow palate

Trigonocephaly, hypertelorism, prominent forehead, long face, micro-gnathia, prominent eyes, upslanting palpebral fissures

High and broad forehead, anteverted nares, hypertelorism

Ears Low-set, cupped Posteriorly rotated Posteriorly rotated Posteriorly rotated Posteriorly rotated, low- Thick ear lobes Low-set, Low-set,

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and overfolded ears set posteriorly rotated posteriorly rotated

Skin Glabellar nevus flammeus, capillary malformations on trunk, neck, and behind ears, deep palmar creases

Glabellar nevus flammeus, deep palmar creases

Glabellar nevus flammeus, capillary malformations on back and neck, deep palmar creases,hypertrichosis

Glabellar nevus flammeus, normal palmar creases

Glabellar nevus flammeus, fetal fingertip pads

Glabellar nevus flammeusHypertrichosisDeep palmar and plantar creases

Glabellar nevus flammeus, capillary malformations on philtrum and neck, deep palmar creases,hypertrichosis

Deep palmar creases, hypertrichosis

Episodes of hypoglycemia

None known Since neonatal period, requiring continuous feeds

Episodic, starting at 2.5 years of age

None known None known Present during the neonatal period

NA NA

Congenital heart disease

ASD, PDA ASD, left ventricular dysfunction

ASD None known None known Thickened pulmonary valve

ASD, VSD PDA, pulmonary artery stenosis

Skeletal/ extremity manifestations

Increased density of alveolar bone, advanced bone age

Kyphosis Scoliosis Multiple fractures, bilateral overlapping 2nd and 4th toes

None known Advanced bone age, thick calvarium, fusion of 2nd and 3rd cervical vertebrae, short metacarpals and distal phalanges

Scoliosis, ulnar deviation of hands, elbow and wrist flexion, scoliosis

Ulnar deviation of hands, clenched hands

Brain MRI findings

White matter volume loss

Increased extra-axial cerebral space, choroid plexus papilloma

Increased extra-axial cerebral space

Increased extra-axial cerebral space

White matter volume loss, ventriculomegaly

Normal Agenesis of corpus callosum, Dandy-Walker malformation

White matter volume loss, Dandy-Walker malformation

ASD: atrial septal defect; DD/ID: developmental delay/intellectual disability; IUGR: intrauterine growth retardation; NA: not available; OFC: occipitofrontal head circumference, PDA: patent ductus arteriosus; SGA: small for gestational age; VSD: ventricular septal defect.* Mutation designations refer to transcript NM_018263.4 and Build GRCh37/hg19.

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